Every year at least two million people are infected with bacteria that cannot be treated with existing antibiotics and 23,000 people die annually as a result of these infections. Our goal is to develop a new, natural product- derived antibiotic that targets Gram positive bacteria, especially MRSAs and other drug-resistant strains. To this end we will use BPL76, a novel ?-keto-macrolide, as the core scaffold for the development of potent analog that are less prone to inducing resistance mechanisms in bacteria than conventional macrolide antibiotics. BPL76, and related compounds were produced in co-culture by two extremophilic fungi isolated from the Berkeley Pit, an acid mine waste lake. BPL76 has demonstrated potency against a panel of Gram- positive bacteria including four MRSA strains with MIC values of 0.8-1.6 g/mL. Specific aim 1: To develop more potent macrolide lactone antibiotics through synthetic modifications of natural-product lead compounds. We plan to generate at least 25-30 well-characterized new analogs to improve potency and selectivity towards both Gram-positive bacteria (and when possible, against, Gram-negative bacteria), and obtain a panel of compounds (lead plus backups) to progress in development towards animal studies. We plan to generate this limited, focused library to address specific SAR hypotheses. Specific aim 2: To identify mechanism of action (MOA) of ?-keto-macrolide antibiotics. Preliminary studies have shown that unlike related macrolides, the MOA of BPL76 does not involve the inhibition of protein synthesis. We will direct studies to determine the actual MOA. Specific aim 3: To conduct Drug Metabolism/Pharmacokinetic (DMPK) and efficacy studies to ascertain resistance, metabolic stability and druggability of ?-keto-macrolides. We expect that 3-5 of the most active analogs synthesized in this study will demonstrate excellent potency and selectivity. This small cohort of select compounds will be evaluated to determine the suitability of the ?-keto- macrolide class as drugs and the likelihood for successful further development. This work is not intended to be a comprehensive PK study but an initial screening to demonstrate that there are no critical PK issues with the compound class. Relevance to public health: if the aims of this project are met, we will have a new, potent macrolide class od antibiotics to help treat Gram-positive infections, especially those caused by drug-resistant bacteria,